Protein Engineering for Biochemical Interrogation and System Design

Abstract

Proteins are intimately involved in almost every cellular phenomenon, from life to death. Understanding the interactions of proteins with each other and other macromolecules and the ability to rationally redesign them to improve their activities or control their function are of considerable current interest. Split-protein methodologies provide an avenue for achieving many of these goals. Since the original discovery of conditionally activated split-ubiquitin, the field has grown exponentially to include the activities of over a dozen different proteins. The flexibility of the systems has resulted in their use across a wide spectrum, both literally and figuratively, to primarily screen, visualize and quantitate macromolecular interactions in a variety of biological systems. In another arena, there is significant interest the apoptosis-regulating proteins: the Bcl-2 family. These proteins are found in many cell types and control, through expression levels as well as other mechanisms, the apoptotic state of a protein as governed by intrinsic death signals generated from such sources as DNA damage and viral infection. The apoptotic function of these proteins are mainly governed by a single type of interaction: the helix:receptor binding of the BH3-Only helices to the anti-apoptotic receptor proteins. While this often promiscuous helix:receptor interaction has received much scrutiny, the nature of the anti-apoptotic binding pocket, especially with regard to the specific residues that govern the interaction, has been lacking. With the high sensitivity and rapid analysis platform afforded by the cell-free split-luciferase analysis methodology, we devised and carried out the first systematic and large scale alanine mutagenesis of all five major anti-apoptotic members of the Bcl-2 family, validated these results both with biophysical methods as well as correlation with previous studies. Our results help explain how different receptors can bind a wide range of helices and also uncovered details regarding binding that are not possible with structural or computational analysis alone. In a second area of research, we have utilized the interaction of BH3 helices and their receptors for designing small molecule controlled protein kinases and phosphatases. In this protein design area, BH3-Only helices were inserted using a knowledge based approach into particular loops within both a protein kinase and a protein phosphatase. The BH3-Only helix interaction with added receptors, such as Bcl-xL provided an allosteric switch for turning-off the activity of the helix-inserted enzymes. The activity of the enzymes could then be turned-on by the addition of a cell-permeable small molecule that is known to bind the receptor. This plug-and-play design was demonstrated to be successful for two very different enzyme classes and likely provides a general and tunable biological element for controlling the activity of one or more proteins and enzymes in a biochemical networks.